IgE antibodies for the inhibition of tumor metastasis

ABSTRACT

The present invention provides novel IgE antibodies useful for inhibiting or preventing metastatic cancer. Also provided are methods to inhibit tumor metastasis by modulating the activity of at least one non-tumor cell, treating a patient to inhibit or prevent tumor metastases of a primary solid tumor, treating metastatic carcinoma, reducing metastasis of carcinoma cells, and reducing the growth kinetics of a primary solid tumor or a metastasized cell or tumor.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/834,169, filed on Jun. 12, 2013. The entire teachings of the aboveapplication are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 30, 2013, isnamed 4173.3003 US1SeqList.txt and is 3,816 bytes in size.

GOVERNMENT SUPPORT

This invention was made with government support under contract CA111639awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

IgE antibodies mediate allergic and asthmatic reactions, characterizedby immediate hypersensitivity and an inflammatory delayed-type responsesrequiring the recruitment of effector cells. IgE antibodies aretransported from the peripheral circulation into tissues, where they canbind allergic effector cells such as mast cells, basophils, eosinophils,dendritic cells, Langerhans cells, monocytes, and macrophages via threetypes of Fc receptors: FcεRI (or high-affinity FcεR) (K_(a)=10¹¹ M⁻¹),FcεRII (or low-affinity FcεR, CD23) (K_(a)<10⁸ M⁻¹), and galectin-3.Unlike antibodies of the IgG class, IgE binds to its FcR with extremelyhigh affinity, which in the case of FcεRI is about three orders ofmagnitude higher than that of IgG for the FcRs (FcγRI-III) and in thecase of FcεRII is as high as that of IgG for its high affinity FcγRI(Gould, H J, et al., Annu. Rev. Immunol., 21: 579-628. (2003); Gounni, AS, et al., Nature, 367: 183-186 (1994); Kinet, J P, Annu. Rev. Immunol.,17: 931-72:931-972 (1999) and Ravetch J V, and Kinet J P, Annu. Rev.Immunol., 9: 457-492 (1991)).

The newly arising field of AllergoOncology is based upon observationsand studies suggesting an inverse correlation between IgE-mediatedallergy and cancer (Turner M C, et al., Int. J. Cancer, 118: 3124-3132(2006); Wang H & Diepgen T L, Allergy 60: 1098-1111, (2005); Turner M C,et al., Am. J. Epidemiol., 162: 212-221 (2005); Wang H, et al., Int. J.Cancer, 119: 695-701 (2006); Mills P K, et al., Am. J. Epidemiol., 136:287-295 (1992); Wiemels J L, et al., Cancer Res., 64: 8468-8473 (2004);Dodig S., et al., Acta Pharm., 55: 123-138 (2005); and Wrensch M., etal., Cancer Res., 66: 4531-4541 (2006)). As a result, researchers inthis field are exploring the therapeutic potential of the IgE antibodyclass in the prevention and treatment of certain cancers, under thepremise that immune responses originally developed as adaptive responsesto microbial/parasitic infection might be useful when directed againstmalignancy.

The application of IgE for the therapy of cancer was pioneered by Nagyet al. (Nagy, E., et al., Cancer Immunol. Immunother., 34: 63-69(1991)), who developed a murine IgE monoclonal antibody specific for themajor envelope glycoprotein (gp36) of mouse mammary tumor virus (MMTV)and demonstrated significant anti-tumor activity in C3H/HeJ mice bearinga syngeneic MMTV-secreting mammary adenocarcinoma (H2712) (Nagy, E., etal., Cancer Immunol. Immunother., 34: 63-69 (1991)). Kershaw et al.(Kershaw, M H, et al., Oncol. Res., 10: 133-142 (1998)) developed amurine monoclonal IgE named 30.6, specific for an antigenic determinantexpressed on the surface of colorectal adenocarcinoma cells. Mouse IgE30.6 inhibited the growth of established human colorectal carcinoma COLO205 cells growing subcutaneously in severe combined immune deficient(SCID) mice, although this effect was transient. By contrast, a mouseIgG 30.6 did not show anti-tumor effects. The mouse IgE specific effectwas attributed to the interaction of the antibody with FcεR bearingeffector cells since the activity was specifically abrogated by prioradministration of a nonspecific mouse IgE (Kershaw, M H, et al., Oncol.Res., 10: 133-142 (1998)). Gould et al. developed a mouse/human chimericIgE (MOv18-IgE) and IgG MOv18 (IgG1) specific for the ovarian cancertumor-associated antigen folate binding protein (FBP). The protectiveactivities of MOv18-IgE and MOv18-IgG1 were compared in a SCID mousexenograft model of human ovarian carcinoma (IGROV1). The beneficialeffects of MOv18-IgE were greater and of longer duration than those ofMOv18-IgG1 demonstrating the superior anti-tumor effects of IgEantibodies (Gould, H J, et al., Eur. J. Immunol., 29: 3527-3537 (1999)).

Recently Karagiannis et al. demonstrated monocyte-mediated IgE-dependenttumor cell killing by two distinct pathways, ADCC (antibody-dependentcell-mediated cytotoxicity) and ADCP (antibody-dependent cell-mediatedphagocytosis), mediated through FcεRI and FcεRII (Karagiannis, S N, etal., Cancer Immunol. Immunother., 57: 247-263 (2008) and Karagiannis, SN, et al., J. Immunol., 179: 2832-2843 (2007)). This group also usedthis assay system to demonstrate that anti-Her2 IgE can activatemonocytes to kill tumor cells in vitro via ADCC (Karragiannis P., etal., Cancer Immunol. Immunother., 58: 915-930 (2009) Published on-line22 Oct. 2008). Additional examples include Fu, et al. (Clin. Exp.Immunol., 153:401-409, 2008) who demonstrated that antibodies of the IgEclass isolated from pancreatic cancer patients mediate ADCC againstcancer cells, and Spillner et al. (Cancer Immunol. Immunother., 61:1565-1573 (2012) who showed using monocytes, that cytotoxicity againstthe human epithelial carcinoma cell line A431 was increased up to 95%with anti-EGFR IgE when compared with anti-EGFR IgG in vitro.

In spite of the encouraging findings in the emerging field ofAllergoOncology, a strong need continues for the development of noveltherapeutics in the treatment of metastatic cancer.

SUMMARY OF THE INVENTION

The present invention provides novel IgE antibodies useful forinhibiting tumor growth and metastases. Provided are therapeuticmonoclonal IgE antibodies comprising the human epsilon constant regionand variable regions comprising the binding specificity for thetumor-associated antigen (TAA). Also provided are methods ofreprogramming the activity of at least one host-derived, non-tumor celllocated in the microenvironment of a primary solid tumor, therebyreversing the ability of the non-tumor cell to promote tumor growth andmetastases. A method for treating metastatic carcinoma, for inhibitingthe primary tumor from giving rise to metastases and for reducing thegrowth kinetics of a primary or metastatic tumor in a patient areprovided.

In one embodiment the invention provides a method for reprogramming, theactivity of at least one host-derived, non-tumor cell located in themicroenvironment of a solid tumor in a patient wherein the ability ofthe non-tumor cell to mediate metastases of the tumor is inhibited,comprising the step of administering an IgE antibody specific for atumor-associated antigen, wherein the antibody forms a ternary complexwithin the microenvironment of the tumor said ternary complex beingcomprised of the IgE antibody, the tumor-associated antigen, and ahost-derived, non-tumor cell endogenous to the tumor environment whereinthe antibody specifically binds to the tumor-associated antigen and anantibody receptor specific for IgE located on the surface of thehost-derived non-tumor cell.

In another embodiment the invention provides a method for inhibitingmetastasis of a solid tumor in a patient, the method comprisingadministering to the patient, an IgE antibody capable of forming aternary complex within the microenvironment of the tumor, said ternarycomplex being comprised of the IgE antibody specific for atumor-associated antigen, the tumor associated antigen and ahost-derived, non-tumor cell endogenous to the tumor microenvironment,wherein the antibody specifically binds to the tumor-associated antigenand an antibody receptor specific for IgE located on the surface of thehost-derived non-tumor cell and wherein upon the formation of theternary complex, the ability of the non-tumor cell to mediate metastasesof the tumor is inhibited.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Construction and antigen specificity of chimeric IgE antibodies.(A) Graphical representations of the vector constructs. The parentvectors were pcDNA 3.1/neomycin (pEpsilon I), pEF6/blastacidin (pEpsilonII), pcDNA 3.1/Zeocin (pKappa I), and pcDNA 3.1/Hygromycin (pKappa II).Heavy and light chain variable region genes were cloned from thehybridomas in Table 1, and inserted upstream of the human epsilon orhuman kappa constant region genes. (B) SDS-PAGE analysis. Chimeric IgEpurified from transfected CHO-K1 clones, and control human IgE frommyeloma cell line SKO-007 were separated by electrophoresis on a 4-12%gradient acrylamide gel and stained with Coomassie blue. (C) Analysis ofantibody specificities. Histogram overlays depict FACS analysis of IgEbinding to cell lines transfected with cognate antigen, compared tountransfected cell lines. 1F5.hIgE (anti-hCD20) bound A20 cellstransfected with human CD20 cDNA, while 3C6.hIgE (anti-hMUC1, partiallyglycosylated form) detected human MUC1 on the mouse breast carcinomacell line 4T-1 transfected with the hMUC1 cDNA. Line graph shows ELISAdetection of a synthetic hMUC1 tandem repeat peptide (50mer) by 4H5.hIgE(anti-hMUC1 protein backbone). The control peptide was derived from thesecond extracellular loop of hCD20 (43mer).

FIG. 2. Mast cell and eosinophil-mediated tumor cytotoxicity in vitro.Histograms show % PI⁺CFSE⁺ tumor cells. OCI-Ly8 human B cell lymphomacells (hCD20⁺) were labeled with 10⁻⁵ mM CFSE for 15 mins. 1×10⁵ labeledcells were mixed with unstained CBMCs and 2.5 μg/ml IgE (A and B) orcord blood-derived eosinophils (CBEos) and 5 μg/ml IgE (C and D), thenincubated at 37° C. for 24 h. The cell mixture was stained withpropidium iodide (PI) before flow cytometric analysis. The percentage ofPI⁺ cells in the CFSE^(hi) fraction represents total tumor cytotoxicity,and results are shown in histogram form. (A) CBMC and hIgE-mediatedtumor cytotoxicity at different effector:target ratios. (B) 2 μg/mlblocking antibodies or isotype control were added (E:T ratio 4:1). (C)CBEos and hIgE-mediated tumor cytotoxicity at different effector:targetratios. (D) 2 μg/ml blocking antibodies or 10 U/ml heparin was added.(E:T ratio 2.5:1). Also shown here are data for tumor death induced byantibodies alone (i.e., in the absence of effector cells). Results shownare mean±SD of one representative experiment. Student's t test *,p<0.05; **, p<0.01; ***, p<0.005.

FIG. 3. Tumor-specific IgE inhibits tumor growth in vivo. 10⁵ 4T1.hMUC1tumor cells were inoculated s.c. into the flanks of hFcεRI mice at d0.20 μg control or 3C6.hIgE (anti-hMUC1) was administered at d1, 2, 3, 4,and 5. 2-dimensional caliper measurements were taken until tumorsexceeded 300 mm² in area. (A) Graph of average tumor size against time.Error bars represent mean±SD of each group. Number of surviving/totalmice per group at the last time point is indicated in brackets. Theanti-hMUC1 group is significantly different from the control group (2way ANOVA, p<0.001). (B and C) Tumors were harvested from surviving miceat d34 from the experiment shown in (A), sectioned, and stained for mastcells. Mast cells were mostly absent in the core of the tumors and inperi-tumor regions. Mast cells that were present (red arrows) were notdegranulated (B). Only one tumor from a mouse in the 3C6.hIgE treatmentgroup contained a region that was infiltrated with mast cells, whichshowed evidence of degranulation (C).

FIG. 4. Mouse IgE specific for hMUC1 mediates complete tumor rejectionin FcεRI. Four different combinations of 4T1 tumor cells transfectedwith human MUC1, anti-hMUC1 IgE (3C6.mIgE) and/or cytokines (MCP-1,IL-5) were inoculated s.c. into the flanks of hFcεRI Tg⁺ mice at d0. 10⁵total cells were administered per mouse (n=4 per group). (A) Descriptionof the four experimental groups. (B) The experiment was designed to testthe interaction of the IgE antibody with the cognate antigen (MUC1) andthe myeloid cells attracted by two cytokines examined. The cells weremixed immediately before injection and then injected subcutaneously intothe flanks of FcεRI KO/Tg mice (100,000 cells/mouse). Tumor growth wasmonitored by two dimensional caliper growth. Progressive growth wasobserved in each group, except for group 4, where the tumors weretransiently palpable, then permanently regressed. Data shown isrepresentative of two separate experiments. Error bars represent averagetumor size±SD.

FIG. 5. Mouse IgE specific for hMUC1 mediates complete tumor rejectionin FIεRI KO/Tg mice. Individual growth curves and averages of tumormeasurements from a repeat experiment of the one shown in FIG. 6. 4T1tumor cells expressing either 3C6.mIgE or cytokines, were mixed as shownin FIG. 6A. Data are from individual tumor measurements (A,C) or theaverages of the tumor volumes (B,D). Note that in the repeat experiment,there was one group 4 tumor that exhibited growth, albeit at greatlyreduced growth kinetics.

FIG. 6. Mouse IgE specific for hMUC1 is unable to mediate complete tumorrejection in wild type mice, but reveals the ability to preventmetastases. Groups of 4T1 tumor cells expressing either 3C6.hIgE orcytokines were mixed as shown in FIG. 6A, and injected into Balb/c mice.Shown are the individual tumor growth curves (A,B), and averages of thegroup (C,D). Group 4 tumors, which did not grow in the FcεRI KO/Tg mice,grew in the wild type mice, although with greatly reduced growthkinetics. Note that the group 3 tumors (4T1/hMUC1 and cytokines alone)exhibited intermediate growth kinetics. Three FcεRI KO/Tg transgenicmice were included in this experiment as a positive control, and allthree failed to demonstrate any tumor growth when injected with group 4tumors (panel D, □—□). The reduced growth kinetics of the group 3 tumors(MUC1 and cytokines only) is likely from the fact that these micedevelop metastases early in the course of tumor development, and beginto lose weight by day 10 (FIG. 7). The mice bearing group 4 tumors(3C6-mIgE+MUC1+cytokines), demonstrate no weight loss, or other overtsigns of metastases, despite having small tumors in their flanks.

FIG. 7. Weight loss kinetics in groups 3 and 4 mice. Wild-Type miceinjected with groups 3 and 4 tumors were weighed every 5 days and theaverage and standard deviations were calculated. Note that significantdifferences in average weight between the two groups were detectable byday 10. Mice in group 3 rapidly lost weight, developed ascites, and gotruffled fur. None of these signs of systemic metastases developed inwild type mice bearing group 4 tumors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel IgE antibodies useful forinhibiting or preventing tumor growth and metastasis. Tumor metastasisis, in part, driven by the interplay of cytokines and bone marrow(myeloid) derived cells of the innate immune system present within thetumor microenvironment. Local expression of cytokines by tumor cellsattracts myeloid suppressor cells, for example, into the tumormicroenvironment, which in turn results in the release of additionalcytokines by these cells. The cytokine milieu within the tumormicroenvironment thus becomes complex, with dozens if not hundreds ofcytokines and chemokines contributing to cancer-associated inflammation.

Tumor-associated myeloid-derived cells such as macrophages and mastcells that accumulate in the tumor microenvironment appear to beassociated with tumor progression (DePalma et al., Cancer Cell 23(3):277-286 (2013); Dalton et al., Cancer Immunol Immunother DOI10.1007/s00262-012-1246-0 Published online Apr. 18, 2012). Thisassociation is modulated by the complex cytokine milieu found within thetumor microenvironment. For example, mammary carcinoma metastasis isenhanced by macrophages in the presence of type 2 cytokines (Ruffell Bet al., Trends Immunol. 33: 119 (2012)). Also, in response to cytokines,other cells such as monocytes, granulocytes, and mast cells secreteproteolytic enzymes that modify the extracellular matrix (ECM),resulting in release of ECM-bound growth factors that facilitatemetastasis (Hanahan D & Coussens L M, Cancer Cell 21: 309 (2012); Lu P,et al., Cold Spring Harb. Perspect. Biol., 3: a005058 (2011)). Itappears, therefore, that “chronically activated myeloid cells inneoplastic tissues support many of the hallmarks of cancer” (Coussens LM et al., Science 339: 286-291 (2013)); (Hanahan D & Coussens L M,Cancer Cell 21: 309 (2012)).

The inventors have unexpectedly discovered that the antibodies of theinvention can inhibit metastases of primary solid tumors as well assecondary tumors and higher. Without being bound by theory, the IgEantibodies in accordance with the invention can reprogram host-derivednon-tumor cells in the tumor microenvironment of a primary tumor suchthat tumor metastasis is inhibited or prevented from occurring. Theinvention is unique and unexpected in that it provides for modulatingthe behavior of a tumor cell through an intermediary, host derived cell.The end result is that a primary tumor will not metastasize. This is instark contrast to heavily documented and well-known aspects ofantibody-based cancer therapy wherein antibody-dependent cell-mediatedcytoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis(ADCP) are employed in the treatment of cancer to kill cancer cellsdirectly Fu, et al. Clin. Exp. Immunol., 153:401-409, 2008; KaragiannisP., et al., Cancer Immunol. Immunother., 58: 915-930 (2009) Publishedon-line 22 Oct. 2008; Karagiannis, S N, et al., Cancer Immunol.Immunother., 57: 247-263 (2008); and Karagiannis, S N, et al., J.Immunol., 179: 2832-2843 (2007)).

In one embodiment of the invention, reprogramming of host-derived,non-tumor cells such as myeloid suppressor cells, occurs within thetumor microenvironment of a primary solid tumor. The reprogramming ofthe host-derived, non-tumor cells is mediated, for example, by cytokinesand tumor-specific IgE antibodies within said tumor microenvironmentafter an effective amount of a tumor-specific IgE antibody isadministered to the subject. In one embodiment, the reprogrammingcomprises the formation of a ternary complex within the tumormicroenvironment. In one embodiment this ternary complex formed withinthe tumor microenvironment is comprised of an IgE antibody, a cellbearing the tumor-associated antigen on its surface such as a tumor cellor a soluble tumor-associated antigen, and a host derived, non-tumorcell. The antibody specifically binds to the tumor-associated antigenand an antibody receptor located on the surface of the host-derivednon-tumor cell, after an effective amount of the antibody isadministered to the subject.

The term “tumor microenvironment” or “tumor stroma” means the tissues,cells, molecules, and blood vessels that surround and feed a tumor cell.A tumor's microenvironment is dynamic and a tumor can change itsmicroenvironment, and the microenvironment can affect how a tumor growsand spread.

In one embodiment the host-derived non-tumor cell (or cells) is anendogenous resident of the tumor microenvironment. As used herein theterm “endogenous to the tumor microenvironment” which may be usedinterchangeably with the term “endogenous to the tumor stroma” means oneor more cells of a dynamic compartment of cell types that intercalate orsurround the tumor nest and includes, but is not limited to, connectivetissue, vasculature, and inflammatory cells that will vary from time totime, but that also include tumor cells themselves. Other host-derived,non-tumor cells of the tumor stroma include fibroblasts and vascularcells, including, but not limited to, cells of mesenchymal origin (suchas fibroblasts, vascular progenitor cells, endothelial cells, adipocytesand their precursors and vascular endothelial cells) and myeloid-derivedcells of varying phenotypes including, but not limited to, mast cell,basophil, monocyte macrophage, eosinophil, neutrophil, dendritic cells,Langerhan's cells, platelets, and their progenitors and alsoinfiltrating lymphocytes including CD4 and CD8 T cells and B cells.Allergic effector cells and/or myeloid suppressor cells are alsocollectively referred to herein as myeloid-derived cells or cells ofmyeloid lineage.

In a preferred embodiment, the antibody comprises human Fc epsilonconstant regions. In one preferred embodiment, the IgE antibody is anantibody specific to CA125, folate binding protein (FBP), HER2/neu,MUC1, and PSA.

As used herein, a “subject” is a human patient or other animal such asanother mammal with functional mast cells, basophils, neutrophils,eosinophils, monocytes, macrophages, dendritic cells, and Langerhanscells. In humans, all of these cells express the high affinity receptorfor IgE (FIεRI) for the administered IgE antibody of the invention.

In some instances the IgE antibodies of the invention can be used toinhibit spread from a primary tumor to a site that is distinct from thesite of the primary tumor also known as metastases. These additionaltumor sites are sometimes referred to as secondary, tertiary,quaternary, or higher tumor sites when they are the result of metastasesfrom a primary tumor site. Primary tumors release circulating cancercells that first must set up microenvironments within various tissuesand organs such as liver, lungs, and bone. These eventually give rise toclinically and radiographically apparent metastases. Unlike IgGantibodies, which primarily stay within the vascular compartment, IgEantibodies migrate out of the vascular space to every tissue in thebody.

In one embodiment, the reprogramming of host-derived, non-tumor cellsendogenous to the tumor microenvironment occurs within themicroenvironment of a secondary, tertiary, quaternary, or higher tumorsite formed by circulating cancer cells. In a second embodiment, cellreprogramming is mediated by tumor-specific antibodies within saidsecondary, tertiary, quaternary, or higher tumor microenvironment,wherein an effective amount of a tumor-specific antibody is administeredto the subject.

In one embodiment, the host-derived non-tumor cell is a myeloid derivedsuppressor cell that is endogenous to the tumor microenvironment. In apreferred embodiment, myeloid derived suppressor cells are independentlyselected from mast cells, basophils, neutrophils, eosinophils,monocytes, macrophages, dendritic cells, and Langerhans cells. In oneembodiment, the host-derived non-tumor cell is of mesenchymal origin andare independently selected from fibroblasts, vascular progenitor cells,endothelial cells, adipocytes and their precursors.

The inventors have also discovered that the antibodies of the inventioncan mediate tumor rejection. The effect was shown to be mediated bymyeloid-derived cells activated by IgE and tumor-associated cell surfaceantigens. The killing of tumors was the result of local effects of IgEantibodies, the presence of myeloid-derived cells in the environment,and cytokines chemotactic for macrophages, eosinophils. In oneembodiment, the IgE antibodies of the invention can be used to inducetumor cell death at secondary, tertiary, quaternary, or highermicroenvironment sites after metastasis has occurred. In a secondembodiment, tumor cell death at secondary, tertiary, quaternary, orhigher microenvironment sites is mediated by altering the pattern ofcytokines present therein and alteration in the activation state ofnon-tumor cells, all in response to tumor-specific antibodies withinsaid microenvironments. In a preferred embodiment, tumor cell deathrequires ternary complex formation within said microenvironments, theternary complex being comprised of an IgE antibody, a tumor cell bearingthe target antigen, and a non-tumor cell. Specifically the IgE antibodyspecifically binds to an antigen located on the surface of the tumorcell and an antibody receptor located on the surface of the non-tumorcell, wherein an effective amount of the antibody is administered to thesubject. In another embodiment, the non-tumor cell is myeloid derivedsuppressor cell. In a preferred embodiment, the myeloid derivedsuppressor cells is independently selected from mast cells, basophils,neutrophils, eosinophils, monocytes, macrophages, dendritic cells, andLangerhans cells. In a preferred embodiment, the IgE antibody isanti-MUC1 IgE.

The inventors have further discovered that the antibodies of theinvention can be used to reduce the growth kinetics of a primary solidtumor or a metastasized cell or tumor in a patient. In one embodiment,the reprogramming of myeloid derived cells occurs within the tumormicroenvironment of a primary solid tumor or a metastasized group ofcells. In a second embodiment, the reprogramming is mediated bytumor-specific antibodies within said tumor microenvironment, wherein aneffective amount of a tumor-specific antibody is administered to thesubject. In a preferred embodiment, the reprogramming requires ternarycomplex formation within the microenvironment of a solid tumor ormetastasized cells or tumor, said ternary complex being comprised of anantibody, a tumor cell of the solid or metastasized cell or tumor, and anon-tumor cell, wherein the antibody specifically binds to an antigenlocated on the surface of the tumor cell and an antibody receptorlocated on the surface of the non-tumor cell, and wherein an effectiveamount of the antibody is administered to the subject. In anotherembodiment, the non-tumor cell is a myeloid-derived cell. In a preferredembodiment, the myeloid-derived cell is of myeloid lineage independentlyselected from mast cells, myeloblasts, basophils, neutrophils,eosinophils, monocytes, macrophages, dendritic cells, and Langerhanscells. In a preferred embodiment, the antibody is IgE. In a morepreferred embodiment, the IgE antibody is anti-MUC1 IgE. As used herein,a “subject” is a human patient or other animal with functional mastcells, myeloblasts, basophils, neutrophils, eosinophils, monocytes,macrophages, dendritic cells, and Langerhans cells with receptoraffinity for the administered IgE antibody of the invention.

A reduction in the growth kinetics of a primary solid tumor or ametastasized cell or tumor as used herein is defined to mean that whichis as understood in the art. For example, a reduction in growth kineticsmeans a reduction in the exponential growth, specific growth rate, ordoubling time of a primary solid tumor, metastasized cell, ormetastasized tumor relative to the exponential growth, specific growthrate, or doubling time normally observed in vivo or in vitro for a giventumor type.

A “therapeutic IgE antibody” of the invention (also referred to hereinas a “monoclonal IgE antibody of the invention”) is a monoclonalantibody that comprises the human epsilon (ε) constant region and alsocomprises variable regions comprising at least one antigen bindingregion specific for a tumor-associated antigen (TAA) that is a cellsurface antigen or a soluble cancer antigen located in the tumormicroenvironment or otherwise in close proximity to the tumor beingtreated. It is believed that the therapeutic dosage of the IgE antibodyof the invention will be much lower than that associated with IgGclasses of antibody therapy against cancer (e.g. trastuzumab(HERCEPTIN®) and rituximab (RITUXAN®)) not only due to the high affinityof IgE to the FcεRI but also because the methods of the invention thatcomprise reprogramming of one or more host-derived non-tumor cellsendogenous to the tumor microenvironment appears to have a cascadingeffect within the tumor environment which facilitates inhibition ofmetastases of a solid tumor. This cascading effect means that IgEantibody in accordance with the invention need not be administered tomediate pharmacologic effects to the target antigens and thus forexample there is no need to saturate CD20 or HER2/neu receptors, forexample, as is necessary with conventional IgG based monoclonal cancertherapy.

The term “tumor-associated antigen” (TAA) as used herein can be any typeof cancer antigen that may be associated with a tumor as is known in theart and includes antigens found on the cell surface of cells includingtumor cells as well as soluble cancer antigens. Such antigens include,but are not limited to cancer-associated fibroblasts (CAFs), tumorendothelial cells (TEC) and tumor-associated macrophages (TAM). Examplesof cancer-associated fibroblasts (CAFs) include but are not limited to:carbonic anhydrase IX (CAIX); fibroblast activation protein alpha(FAPα); and matrix metalloproteinases (MMPs) including MMP-2 and MMP-9.Examples of Tumor endothelial cell (TECs) target antigens include, butare not limited to vascular endothelial growth factor (VEGF) includingVEGFR-1, 2, and 3; CD-105 (endoglin), tumor endothelia markers (TEMs)including TEM1 and TEM8; MMP-2; Survivin; and prostate-specific membraneantigen (PMSA). Examples of tumor associated macrophage antigensinclude, but are not limited to: CD105; MMP-9; VEGFR-1, 2, 3 and TEM8.

In one embodiment, the therapeutic IgE antibody may be specific forcancer antigens located on tumor cells, for example, VEGFR-2, MMPs,Survivin, TEM8 and PMSA. The cancer antigen may be an epithelial cancerantigen, (e.g., breast, gastrointestinal, lung), a prostate specificcancer antigen (PSA) or prostate specific membrane antigen (PSMA), abladder cancer antigen, a lung (e.g., small cell lung) cancer antigen, acolon cancer antigen, an ovarian cancer antigen, a brain cancer antigen,a gastric cancer antigen, a renal cell carcinoma antigen, a pancreaticcancer antigen, a liver cancer antigen, an esophageal cancer antigen, ahead and neck cancer antigen, or a colorectal cancer antigen. A cancerantigen can also be a lymphoma antigen (e.g., non-Hodgkin's lymphoma orHodgkin's lymphoma), a B-cell lymphoma cancer antigen, a leukemiaantigen, a myeloma (i.e., multiple myeloma or plasma cell myeloma)antigen, an acute lymphoblastic leukemia antigen, a chronic myeloidleukemia antigen, or an acute myelogenous leukemia antigen.

Other cancer antigens include but are not limited to mucin-1 protein orpeptide (MUC-1) that is found on all human adenocarcinomas: pancreas,colon, breast, ovarian, lung, prostate, head and neck, includingmultiple myelomas and some B cell lymphomas; mutated B-Raf antigen,which is associated with melanoma and colon cancer; human epidermalgrowth factor receptor-2 (HER-2/neu) antigen; epidermal growth factorreceptor (EGFR) antigen associated lung cancer, head and neck cancer,colon cancer, colorectal cancer, breast cancer, prostate cancer, gastriccancer, ovarian cancer, brain cancer and bladder cancer;prostate-specific antigen (PSA) and/or prostate-specific membraneantigen (PSMA) that are prevalently expressed in androgen-independentprostate cancers; gp-100 (Glycoprotein 100) associated with melanomacarcinoembryonic (CEA) antigen; carbohydrate antigen 19.9 (CA 19.9)related to the Lewis A blood group substance and is associated withcolorectal cancers; and a melanoma cancer antigen such as MART-1.

Other antigens include mesothelin, folate binding protein (FBP),carbohydrate antigen 125 (CA-125) and melanoma associated antigens suchas NYESO 1.

In one embodiment, the cancer antigen is a soluble cancer antigen. In apreferred embodiment the tumor-associated target antigen is a cellsurface antigen located on the surface of a tumor cells. In onepreferred embodiment the tumor associated antigen is selected fromCA125, folate binding protein (FBP), HER2/neu, MUC1, and PSA.

The terms “monoclonal antibody” or “monoclonal antibodies” as usedherein refer to a preparation of antibodies of single molecularcomposition. A monoclonal antibody composition displays a single bindingspecificity and affinity for a particular epitope. The monoclonalantibodies of the present invention are preferably chimeric, humanized,or fully human in order to bind human Fc epsilon receptors when thesubject host is a human. Humanized and fully human antibodies are alsouseful in reducing immunogenicity toward the murine components of, forexample, a chimeric antibody, when the host subject is human. Monoclonalantibodies may be prepared by standard techniques including, but notlimited to, recombinantly and synthetically.

The term “chimeric monoclonal antibody” refers to antibodies displayinga single binding specificity, which have one or more regions derivedfrom one antibody and one or more regions derived from another antibody.In one embodiment of the invention, the constant regions are derivedfrom the human epsilon (ε) constant region (heavy chain) and human kappaor lambda (light chain) constant regions. The variable regions of achimeric IgE monoclonal antibody of the invention are typically ofnon-human origin such as from rodents, for example, mouse (murine),rabbit, rat or hamster.

As used herein, “humanized” monoclonal antibodies comprise constantregions that are derived from human epsilon constant region (heavychain) and human kappa or lambda (light chain) constant regions. Thevariable regions of the antibodies preferably comprise a framework ofhuman origin and antigen binding regions (CDRs) of non-human origin.

Fully human or human-like antibodies may be produced through vaccinationof genetically engineered animals such as mouse lines produced atAbgenix Inc. (Thousand Oaks, Calif.) and MedaRex (Princeton, N.J.) whichcontain the human immunoglobulin genetic repertoire and produce fullyhuman antibodies in response to vaccination. Further, the use of phagedisplay libraries incorporating the coding regions of human variableregions which can be identified and selected in an antigen-screeningassay to produce a human immunoglobulin variable region binding to atarget antigen.

The term “antigen binding region” refers to that portion of an antibodyof the invention which contains the amino acid residues that interactwith an antigen and confer on the antibody its specificity and affinityfor the antigen. The antibody region includes the “framework” amino acidresidues necessary to maintain the proper confirmation of the antigenbinding residues.

An “antigen” is a molecule or portion of a molecule capable of beingbound by an antibody, which is additionally capable of inducing ananimal to produce antibody capable of binding to an epitope of thatantigen. An antigen can have one or more epitopes that are the same ordifferent. In a preferred embodiment, the antibodies of the inventionare specific for a single epitope. In one embodiment, the antigen is acapable of being bound by an IgE antibody of the invention to form animmune complex that in combination with a myeloid effector cell iscapable of reprogramming the tumor microenvironment to inhibit orprevent tumor metastasis. In one embodiment, the antigen, on its own,may not be capable of stimulating an immune response for any number ofreasons, for example, the antigen is a “self” antigen, not normallyrecognized by the immune system as requiring response or the immunesystem has otherwise become tolerant to the antigen and does not mountan immune response. In another embodiment, the antigen is MUC1.

The term “epitope” is meant to refer to that portion of an antigencapable of being recognized by and bound by an antibody at one or moreof the antibody's binding regions. Epitopes generally comprisechemically active surface groupings of molecules such as amino acids orsugar side chains and have specific three dimensional structurecharacteristics as well as specific charge characteristics. In oneembodiment, an epitope of an antigen is a repetitive epitope. In oneembodiment an epitope of an antigen is a non-repetitive epitope.

A “ternary complex” is a complex formed in the microenvironment of atumor comprised of an IgE antibody of the invention, a tumor-associatedtarget antigen, and a host-derived non-tumor cell, wherein the antibodyspecifically binds to a tumor associated antigen and an antibodyreceptor located on the surface of the non-tumor cell.

In one preferred embodiment, the antigen is CA-125, folate bindingprotein (FBP), HER2/neu, MUC1 or PSA. In one embodiment, the non-tumorcell is an effector cell. In one embodiment, the effector cell is anallergic and/or anti-parasitic effector cell of myeloid lineage. In apreferred embodiment, the IgE is anti-MUC1 IgE. In a preferredembodiment, the antibody receptor is FcεRI.

In one embodiment, a ternary complex includes an IgE antibody bound to asoluble cancer antigen and an antibody receptor located on the surfaceof a host-derived non-tumor cell. In a preferred embodiment the ternarycomplex includes an IgE antibody bound to a tumor-associated targetantigen expressed on the surface of the tumor cell and an FcεRI locatedon a host-derived, non-tumor cell endogenous to the tumormicroenvironment.

Methods for raising antibodies, such as murine antibodies to an antigen,and for determining if a selected antibody binds to a unique antigenepitope are well known in the art.

Screening for the desired antibody can be accomplished by techniquesknown in the art, e.g., radioimmunoassay, ELISA (enzyme-linkedimmunosorbant assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitin reactions, immunodiffusion assays, in situimmunoassays (using colloidal gold, enzyme or radioisotope labels, forexample), western blots, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

For preparation of monoclonal antibodies, any technique that providesfor the production of antibody molecules by continuous cell lines inculture may be used (see, e.g., Antibodies—A Laboratory Manual, Harlowand Lane, eds., Cold Spring Harbor Laboratory Press: Cold Spring Harbor,N.Y., 1988). These include but are not limited to the hybridomatechnique originally developed by Kohler and Milstein (1975, Nature256:495-497), as well as the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). In an additional embodiment of the invention,monoclonal antibodies can be produced in germ-free animals utilizingrecent technology (PCT/US90/02545). According to the invention, humanantibodies may be used and can be obtained by using human hybridomas(Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A., 80:2026-2030) or bytransforming human B cells with EBV virus in vitro (Cole et al., 1985,in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).In fact, according to the invention, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., 1984, J.Bacteriol. 159: 870; Neuberger et al., 1984, Nature 312:604-608; Takedaet al., 1985, Nature 314: 452-454) by splicing the genes from a mouseantibody molecule specific for a polypeptide together with genes from ahuman antibody molecule of appropriate biological activity can be used;such antibodies are within the scope of this invention.

In one embodiment, the antibody of the invention is an IgE monoclonalantibody comprising a nucleic acid sequence selected from a heavy chainvariable region encoded by a nucleic acid sequence comprising SEQ ID NO:1; a light chain variable region encoded by a nucleic acid sequencecomprising SEQ ID NO: 2 and any combination thereof and wherein theheavy and or light chain or both is grafted onto human Ig kappa lightchain and epsilon heavy chain genes.

In one embodiment the antibody of the invention is an IgE monoclonalantibody comprising a nucleic acid sequence selected from a heavy chainvariable region encoded by the nucleic acid of SEQ ID NO: 3; a lightchain variable region encoded by the nucleic acid of SEQ ID NO: 4 andany combination thereof and wherein the heavy and or light chain or bothis grafted onto human Ig kappa light chain and epsilon heavy chaingenes.

In one embodiment, the invention provides a monoclonal antibody,3C6.hIgE, comprising variable regions of the light and heavy chain ofIgG cloned from the VU-3C6 hybridoma, and grafted onto human Ig kappalight chain and epsilon heavy chain genes. VU-3C6 targets human mucin 1(hMUC1), a mucin overexpressed on tumors arising from glandularepithelium. In one embodiment, the invention comprises the IgE antibody,4H5hIgE, which is specific to an isoform of MUC1 different from the MUC1isoform that 3C6.hIgE is specific to.

In one embodiment, the antibody of the invention is the monoclonalantibody 3C6.hIgE comprising a heavy chain variable region encoded by anucleic acid sequence comprising SEQ ID NO: 1; a light chain variableregion encoded by a nucleic acid sequence comprising SEQ ID NO: 2.

In one embodiment the antibody of the invention is the monoclonalantibody 4H5hIgE. The antibody 4H5.hIgE has a heavy chain variableregion encoded by the nucleic acid of SEQ ID NO: 3 and a light chainvariable region encoded by the nucleic acid of SEQ ID NO: 4 and graftedonto human Ig kappa light chain and epsilon heavy chain genes.

In one embodiment, the antibody of the invention is an IgE monoclonalantibody specific for an epitope of MUC1. In one embodiment, theantibody of the invention is specific for the epitope of MUC1 comprisingamino acids STAPPAHGVTSAPDTRPAPG [SEQ ID NO: 5] of MUC1. The exactepitope lies in one of the 20 amino acid repeats that characterize theexternal domain of MUC1. In one embodiment, the antibody of theinvention is capable of binding MUC1 at the epitope defined atSTAPPAHGVTSAPDTRPAPG [SEQ ID NO: 5].

In one embodiment, antibodies in accordance with the present inventionare expressed by a positive transfectoma which is identified byenzyme-linked immunosorbent assay (ELISA) and Western Blot. The positivetransfectoma will be cloned by limited dilution for highest productivityand selected for antibody production. As used herein a “transfectoma”includes recombinant eukaryotic host cells expressing the antibody, suchas Chinese hamster ovary (CHO) cells and NS/O myeloma cells. Suchtransfectoma methodology is well known in the art (Morrison, S. (1985)Science, 229:1202). Previously published methodology used to generatemouse/human chimeric or humanized antibodies has yielded the successfulproduction of various human chimeric antibodies or antibody fusionproteins (Helguera G, Penichet M L., Methods Mol. Med.(2005)109:347-74).

In general, chimeric mouse-human monoclonal antibodies (i.e., chimericantibodies) can be produced by recombinant DNA techniques known in theart. For example, a gene encoding the Fc constant region of a murine (orother species) monoclonal antibody molecule is digested with restrictionenzymes to remove the region encoding the murine Fc, and the equivalentportion of a gene encoding a human Fc constant region is substituted.(See Robinson et al., International Patent Publication PCT/US86/02269;Akira, et al., European Patent Application 184,187; Taniguchi, M.,European Patent Application 171, 496; Morrison et al., European PatentApplication 173,494; Neuberger et al., International Application WO86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent Application 125,023; Better et al. (1988 Science,240:1041-1043); Liu et al. (1987) PNAS, 84:3439-3443; Liu et al., 1987,J. Immunol., 139:3521-3526; Sun et al. (1987) PNAS, 84:214-218;Nishimura et al., 1987, Canc. Res., 47:999-1005; Wood et al. (1985)Nature, 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst.,80:1553-1559).

The chimeric antibody can be further humanized by replacing sequences ofthe Fv variable region which are not directly involved in antigenbinding with equivalent sequences from human Fv variable regions.General reviews of humanized chimeric antibodies are provided byMorrison, S. L., 1985, Science, 229:1202-1207 and by Oi et al., 1986,BioTechniques, 4:214. Those methods include isolating, manipulating, andexpressing the nucleic acid sequences that encode all or part ofimmunoglobulin Fv variable regions from at least one of a heavy or lightchain. Sources of such nucleic acid are well known to those skilled inthe art and, for example, may be obtained from 7E3, ananti-GPII_(b)III_(a) antibody producing hybridoma. The recombinant DNAencoding the chimeric antibody, or fragment thereof, can then be clonedinto an appropriate expression vector. Suitable humanized antibodies canalternatively be produced by CDR substitution (U.S. Pat. No. 5,225,539;Jones et al. 1986 Nature, 321:552-525; Verhoeyan et al. 1988 Science,239:1534; and Beidler et al. 1988 J. Immunol., 141:4053-4060).

In one embodiment, the immunogenicity of an IgE monoclonal antibody ofthe invention is reduced as compared to, for example, the parentantibody from which it was derived, using various strategies. Forexample, a chimeric IgE monoclonal antibody of the invention comprisinghuman Fcε constant regions and murine variable regions may be renderedless immunogenic to the human subject by genetically engineeringhumanized antibodies which comprise constant regions that are derivedfrom human Fcε and variable regions that comprise a framework of humanorigin and antigen binding regions of non-human origin that maintain thesame antigen specificity as that of the parent chimeric antibody.Alternatively, fully human or human like antibodies comprising the sameantigen specificity as the parent chimeric IgE monoclonal antibodies mayalso be genetically engineered using known procedures.

Other processes for reducing the immunogenicity of IgE monoclonalantibodies of the invention include, but are not limited to, processessuch as DE-IMMUNIZATION™ (Biovation Ltd., Aberdeen, United Kingdom andMerck KgaA, Darmstadt, Germany). This technology is a process thatidentifies murine epitopes present on murine or chimeric monoclonalantibodies that might cause immunogenicity in humans such as “humananti-mouse antibody” (HAMA) or “human anti-chimeric antibody” (HACA).This process further genetically alters these epitopes to avoid or atleast reduce immunogenicity as compared to antibodies that have not beensubjected to this process.

Other methods of reducing immunogenicity of monoclonal antibodies (Lazaret al., Mol Immunol., 44, 1986-1998 (2007)) identifies framework andantigen binding region peptides or conformational motifs that mayactivate T-helper cells resulting in HAMA or HACA responses. Using thismethod a novel quantitative paradigm is used to determine the“humanness” of murine variable regions and murine regions of low humanidentity can be substituted for regions of higher human identity therebyreducing immunogenicity of the antibody.

As used herein the induction of an IgE-mediated immune response includesone or more of the following:

i) Ternary complex formation within a tumor microenvironment or on atumor cell comprising binding of an IgE antibody to a myeloid-derivedcell and to a tumor-specific antigen such that reprogramming of themyeloid-derived cell inhibits or prevents tumor metastasis;

ii) Hypersensitivity against the antigen/IgE immune complex particularlyin the tumor microenvironment as evidenced by degranulation of mastcells and basophils bound to such immune complex via IgE antibodyreceptors FcεRI and/or FcεRII and the release of histamine, for example;

iii) Direct targeting of tumor cells via ADCC immune responses, ADCPimmune responses or both ADCC and ADCP immune responses against theantigen/IgE immune complex particularly in the tumor microenvironment asevidenced by the stimulation of eosinophils, mast cells, basophils, andother cells to release pro-inflammatory cytokines, proteases andvasoactive lipid mediators (e.g. leukotrienes, prostaglandin D2, andplatelet activating factor when bound to the antigen/IgE immune complexvia IgE antibody receptors FcεRI and FcεRII;

iv) a cellular response as evidenced in part by the production ofT-cells that are specific for the antigen, the antigen/IgE antibodyimmune complex, or a peptide of the antigen complexed with MHC;

v) a Th1/Tc1 immune response in response to challenge with theantigen/IgE antibody immune complex as evidenced, for example, by theproduction of CD8 IFN gamma positive T cells in response to the tumorantigen and tumor;

vi) a humoral response as evidenced by production of antibodies againstthe antigen or the antigen/IgE immune complex.

As used herein, an “effective amount” of an IgE monoclonal antibody ofthe invention is that amount sufficient to recognize and bind theepitope of the TAA that is a cell surface antigen and induce, elicit, orenhance the referenced immune response in accordance with the invention.

The invention also provides a method for inducing an IgE-mediated immuneresponse against a cell surface antigen on a circulating tumor cell in asubject capable of mounting such a response. This comprisesadministering to the subject an effective amount of an IgE monoclonalantibody that specifically binds an epitope of the surface of acirculating tumor cell antigen wherein an IgE mediated immune responseagainst the tumor results.

As used herein the induction of an IgE-mediated immune responsetriggered by a cell surface antigen on a circulating tumor cell, orclinically unapparent metastases is evidenced, in part, by any one ofthe following:

-   -   i) the inhibition of tumor growth and/or the facilitation of        tumor destruction, in whole or in part, resulting from        reprogramming of myeloid derived cells in the tumor        microenvironment;    -   ii) acute inflammation in the tumor environment and subsequent        tumor growth inhibition and or destruction via effector cells        bearing human Fc epsilon receptors able to bind-monoclonal IgE        antibody and direct ADCC immune responses, ADCP immune responses        or both ADCC and ADCP immune responses reactions to the antigen        in the microenvironment;    -   iii) secondary T cell responses evidenced by the appearance of        T-cells displaying specificity for the target tumor antigen or        secondarily additional antigens derived from the tumor and        expressed in the context of MHC on the tumor cell resulting in        tumor growth inhibition or lysis and destruction; or    -   iv) T cell response against the tumor evidenced by the        production of T cells against other antigens associated with        tumor cells that have been lysed as above in (ii).

The invention also provides methods of inducing direct IgE-mediated ADCCimmune responses, ADCP immune responses, or both ADCC and ADCP immuneresponses to a TAA on the surface of a primary tumor, or to a TAA on thesurface of circulating metastasized tumor cell in a subject capable ofmounting such an immune response comprising administering to the subjectan effective amount of an IgE monoclonal antibody that specificallybinds a single epitope of the circulating antigen wherein an IgEmediated ADCC immune response and possibly or optionally an ADCP immuneresponse against the antigen is elicited. In a preferred embodiment, theADCC immune response and possibly or optionally an ADCP immune responseis elicited in the microenvironment of a tumor or tumor cell. In anotherembodiment the ADCC immune response and possibly or optionally an ADCPimmune response is capable of causing the lysing and killing of tumorcells within the tumor microenvironment or at the tumor cell viabystander effects.

The invention also provides a method for the treatment of cancerassociated with the antigen to which the antibody of the invention isspecific, by administering a composition comprising an IgE monoclonalantibody of the invention that specifically binds at least one singleepitope of a tumor-associated antigen. Such cancers include but are notlimited to pancreatic cancer, gastric cancer (cancer of thegastrointestinal tract), colorectal cancer, and lung cancer. Other typesof cancers that may be treated by the methods of the invention includebut are not limited to: osteosarcoma, esophageal cancer, lung cancer,mesotheliona, liver cancer, gastric cancer, pancreatic cancer,colorectal cancer, rectal cancer, colic cancer, ureteral tumor, braintumor, gallbladder cancer, cholangioma, bile duct cancer, renal cancer,breast cancer, urinary bladder cancer, ovarian cancer, uterocervicalcancer, prostatic cancer, thyroid cancer, testicle tumor, Kaposi'ssarcoma, maxillary cancer, tongue cancer, lip cancer, oral cancer,laryngeal cancer, pharyngeal cancer, myosarcoma, skin cancer and thelike.

In one embodiment, the invention provides a method of treating cancersof epithelial origin in a subject by administering to the subject atherapeutic IgE monoclonal antibody of the invention that specificallybinds an epitope of MUC1. In a preferred embodiment, the therapeutic IgEmonoclonal antibody of the invention that binds a single epitope of MUC1inhibits or prevents tumor metastasis by reprogramming the tumormicroenvironment in the primary tumor, or prevents establishment ofclinically or radiographically apparent metastases by interfering withthe establishment of a network of host-derived, myeloid cells thatpromote tumor growth outside of their tissue of origin.

In one embodiment, the term “reprogramming the tumor microenvironment”means the net effect of antigen bound IgE on host-derived, residentmyeloid cells in a tumor, or allergic effector cells subsequently drawninto the tumor, on tumor growth and metastases. Once bound to both theantigen, and the high affinity FceRI on a host derived cell, located inthe region of the tumor microenvironment, the tumor behavior is changedsuch that the tumor will not form metastasis. In one embodiment,reprogramming the tumor microenvironment is facilitated by the formationof a ternary complex. In one embodiment, the ternary complex iscomprised of the IgE antibody, an effector cell, and a tumor-associatedantigen.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of an antibodyof the invention and a pharmaceutically acceptable carrier. In onepreferred embodiment, the pharmaceutical composition comprises atherapeutic IgE monoclonal antibody of the invention that specificallybinds a single epitope of MUC1.

In one embodiment, the term “pharmaceutically acceptable” means approvedby a regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, and more particularly in humans. The term “carrier” refersto a diluent, adjuvant, excipient, or vehicle with which the therapeuticis administered. Such pharmaceutical carriers can be sterile liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the antibody or fragment thereof,preferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In accordance with a method of the invention compositions comprising theIgE monoclonal antibody of the invention may be administered to thepatient by any immunologically suitable route. For example, the antibodymay be introduced into the patient by an intravenous, subcutaneous,intraperitoneal, intrathecal, intravesical, intradermal, intramuscular,or intralymphatic routes. The composition may be in solution, tablet,aerosol, or multi-phase formulation forms. Liposomes, long-circulatingliposomes, immunoliposomes, biodegradable microspheres, micelles, or thelike may also be used as a carrier, vehicle, or delivery system.Furthermore, using ex vivo procedures well known in the art, blood orserum from the patient may be removed from the patient; optionally, itmay be desirable to purify the antigen in the patient's blood; the bloodor serum may then be mixed with a composition that includes a bindingagent according to the invention; and the treated blood or serum isreturned to the patient. The invention should not be limited to anyparticular method of introducing the binding agent into the patient.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where the composition is to be administered by infusion, it canbe dispensed with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the composition is administered byinjection, an ampoule of sterile water for injection or saline can beprovided so that the ingredients may be mixed prior to administration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the composition of the invention which will be effectivein the treatment, inhibition and prevention of tumor metastasisassociated with the antigen to which the antibody of the invention isspecific can be determined by standard clinical techniques. In addition,in vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation willalso depend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test systems.

For the antibodies of the invention, the dosage administered to apatient is typically 0.001 μg/kg to 1 mg/kg of the patient's bodyweight. Preferably, the dosage administered to a patient is between 0.01μg/kg and 0.1 mg/kg of the patient's body weight, more preferably 0.02μg/kg to 20 μg/kg of the patient's body weight. Generally, the IgEmonoclonal antibodies of the invention have a much higher affinity forthe Fcε R (as compared to IgG antibodies, for example) and longerhalf-life within the human body than antibodies from other species.Thus, lower dosages of the antibodies of the invention and less frequentadministration is often possible.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e. combined with other agents. For example acombination therapy can include a composition of the present inventionwith at least one anti-tumor agent, efficacy enhancing agent, and/orsafety enhancing agent.

The pharmaceutical compositions of the present invention have in vitroand in vivo diagnostic and therapeutic utilities. For example, thesemolecules can be administered to cells in culture, e.g., in vitro or exvivo, or in a subject, e.g., in vivo, to treat cancer. As used herein,the term “subject” is intended to include human and non-human animals. Apreferred subject is a human patient with cancer. As used herein theterms “treat” “treating” and “treatment” of cancer includes: inhibitingtumor metastasis in a patient, inhibiting the onset of cancer in apatient; eliminating or reducing tumor burden in a patient; prolongingsurvival in a cancer patient; prolonging the remission period in acancer patient following initial treatment with chemotherapy and/orsurgery; and/or prolonging any period between cancer remission andcancer relapse in a patient.

As used herein “inhibit”, “inhibition” or “inhibiting” in the context ofthe invention means to slow, hinder, restrain reduce or prevent. Forexample, “inhibiting metastasis” of a primary tumor cell as that term isused herein means to slow, hinder, restrain, reduce or prevent theprimary tumor cell from metastasizing.

As used herein, “administering” refers to any action that results inexposing or contacting a composition containing an antibody of theinvention with a pre-determined cell, cells, or tissue, typicallymammalian. As used herein, administering may be conducted in vivo, invitro, or ex vivo. For example, a composition may be administered byinjection or through an endoscope. Administering also includes thedirect application to cells of a composition according to the presentinvention. For example, during the course of surgery, tumor cells may beexposed. In accordance with an embodiment of the invention, theseexposed cells (or tumors) may be exposed directly to a composition ofthe present invention, e.g., by washing or irrigating the surgical siteand/or the cells.

When used for therapy for the treatment of cancer, the antibodies of theinvention are administered to the patient in therapeutically effectiveamounts (i.e. amounts needed to treat clinically apparent tumors, orprevent the appearance of clinically apparent tumor, either at theoriginal site or a distant site, at some time point in the future. Theantibodies of the invention and the pharmaceutical compositionscontaining them will normally be administered parenterally, whenpossible, at the target cell site, or intravenously.

In another embodiment, the IgE antibodies of the invention can beco-administered with a second therapeutic agent, e.g., achemotherapeutic agent. Such therapeutic agents include, among others,anti-neoplastic agents such as doxorubicin, cisplatin, bleomycinsulfate, paclitaxel, carmustine, chlorambucil, and cyclophosphamide.These agents by themselves, are only effective at levels which are toxicor subtoxic to a patient. Furthermore, these agents are thought not tobe curative in common solid tumors which have spread outside of theirtissue of origin. By co-administering tumor specific IgE antibodies andcommon chemotherapy agents, the goal will be to enhance the activity ofthe chemotherapy agent, by preventing the emergence of chemotherapyresistance, the latter being a product of host derived, non-tumor cellsin the microenvironment.

Pharmaceutical compositions of the present invention can include one ormore further chemotherapeutic agents selected from the group consistingof nitrogen mustards (e.g., cyclophosphamide and ifosfamide), aziridines(e.g., thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g.,carmustine and streptozocin), platinum complexes (e.g., carboplatin andcisplatin), non-classical alkylating agents (e.g., dacarbazine andtemozolamide), folate analogs (e.g., methotrexate), purine analogs(e.g., fludarabine and mercaptopurine), adenosine analogs (e.g.,cladribine and pentostatin), pyrimidine analogs (e.g., fluorouracil(alone or in combination with leucovorin) and gemcitabine), substitutedureas (e.g., hydroxyurea), antitumor antibiotics (e.g., bleomycin anddoxorubicin), epipodophyllotoxins (e.g., etoposide and teniposide),microtubule agents (e.g., docetaxel and paclitaxel), camptothecinanalogs (e.g., irinotecan and topotecan), enzymes (e.g., asparaginase),cytokines (e.g., interleukin-2 and interferon-.alpha.), monoclonalantibodies (e.g., trastuzumab and bevacizumab), recombinant toxins andimmunotoxins (e.g., recombinant cholera toxin-B and TP-38), cancer genetherapies, physical therapies (e.g., hyperthermia, radiation therapy,and surgery) and cancer vaccines (e.g., vaccine against telomerase).

The present invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Creation of Chimeric IgE Gene Vectors

Hybridomas VU-3C6 and VU-4H5 were raised against two different isoformsof human mucin 1 (hMUC-1), a mucin overexpressed on tumors arising fromglandular epithelium. Antibody variable gene segments were cloned fromeach hybridoma, and grafted onto human kappa light chain and epsilonheavy chain gene segments using standard procedures. Kappa cDNA fromhuman peripheral lymphocytes was cloned and then compared the sequenceto the data base sequences (e.g. GenBank: J00241.1). The epsilonconstant region cDNA, was cloned from an IgE-expressing hybridoma(SKO-007, ATCC CRL 8033-1) and compared to the genomic sequence in thedata base (e.g. GenBank: J00222.1). The final IgE mouse-human chimericantibodies were designated 3C6.hIgE and 4H5.hIgE. The final plasmids areshown in FIG. 1A.

The 1F5 hybridoma targets human CD20 (hCD20), a pan-B cell marker andimportant therapeutic target for treatment of B cell lymphomas andseveral autoimmune diseases. Antibody variable gene segments were clonedfrom the 1F5 hybridoma, and grafted onto human Ig kappa light chain andepsilon heavy chain genes using standard procedure as described above.The final IgE chimeric antibody was designated 1F5.hIgE. The finalplasmid is shown in FIG. 1A.

The final plasmids were transfected into CHO-K1 cells (American TypeCulture Collection (ATCC), Manassas, Va.) for antibody production andpurification. Human IgE was purified by Fast Protein LiquidChromatography (FPLC), using an anti-hIgE affinity column. FIG. 1B showsthe SDS-PAGE comparison of the purified chimeric IgEs, to a human IgEisotype control (IgE from human myeloma SKO-007, purified in a similarfashion). All three samples of chimeric antibody are pure, with minorsize differences compared to control hIgE (likely due to differentglycosylation patterns). Under reducing conditions, the epsilon heavychain migrates at 75 kDa, unlike the gamma heavy chain, which migratesas a 50 kDa polypeptide.

The purified antibodies were tested for their ability to bind theirrespective native antigen (i.e. CD20 and MUC1). As analyzed via flowcytometry, 1F5.hIgE bound the A20 mouse B cell lymphoma transfected withhuman CD20, but not the wild type cell line (FIG. 1C left panel).Similarly, 3C6.hIgE bound to 4T1 murine breast cancer cells transfectedwith human MUC1, but not to untransfected 4T1 cells (FIG. 1C centerpanel). 4H5.hIgE bound to a 50-mer peptide derived from the tandemrepeat, extracellular domain of human MUC-1, as detected by ELISA, butdid not bind a control peptide (FIG. 1C right panel). Thus, the use ofthe human epsilon and kappa constant regions, as well as the productionand purification protocol, did not affect antigen recognition by thevariable regions of the original mouse antibodies.

Example 2 IgE-Mediated Tumor Cytotoxicity by 1F5.hIgE (Anti-hCD20)

To determine if the anti-hCD20 chimeric antibody 1F5.hIgE had afunctional IgE Fc region, we investigated its ability to activate cordblood derived mast cells (CBMCs). Using interleukin-8 (IL-8) productionas a measure of CBMC activation, we observed that CBMCs pre-coated with1F5.hIgE produced IL-8 in the presence of hCD20-transfected mouse Bcells (A20.hCD20), but not untransfected cells. Similarly, only CBMCscoated with anti-hCD20 IgE (1F5.hIgE) responded to the hCD20⁺ human Bcell OCI-Ly8, while CBMCs coated with a control IgE (SKO) failed toproduce IL-8 under these conditions. These data demonstrate that1F5.hIgE activates mast cells in an antigen-dependent andantigen-specific manner.

Mast Cells and IgE-Mediated Tumor Cytotoxicity

To test for potential cytotoxic effects mediated by tumor specific IgE,we focused on effector cells known to express and respond to the highaffinity IgE receptor, FcεRI. Previous investigators reported tumorcytotoxicity mediated by human monocytes and tumor-specific IgE(Karagiannis et al. (2003). Eur J Immunol 33:1030-1040). We observedsimilar results using U937 monocytes in our system. In this report, wefocus on data obtained using human mast cells and eosinophils; two celltypes involved in the pathogenesis and tissue damage observed in allergyand asthma (Rothenberg et al., 2006 Nat Rev Immunol 8:205-217; Gould andSutton. 2008 Nat Rev Immunol 8:205-217; Tsai et al., 2005 Chem ImmunolAllergy 87:179-197. Mast cells were of particular interest because theyreside in the tissues where tumors arise, express high levels of FcεRI,and when activated trigger a coordinated inflammatory response thatrecruits eosinophils, neutrophils and other effectors that maypotentially mediate tumor regression (Theoharides and Conti, 2004 TrendsImmunol 25:235-241). Previously, mast cells have been shown to exerttumoricidal effects via TNF and the peroxidase system (Henderson et al.,1981 J Exp Med 153:520-533; Benyon et al., 1991 J Immunol 147:2253-2258;Ozdemir, O. 2007 J Immunol Methods 319:98-103).

To test if activated mast cells can directly induce tumor cell death, weprepared purified, cultured mast cells and incubated them with IgE andtumors. Cultured mast cells derived from cord blood (CBMCs) functionallyresemble human mast cells that have been freshly-isolated from tissues(Saito et al., 1996 J Immunol 157:343-350). CBMCs were mixed withOCI-Ly8 B cells, in the presence of anti-CD20 (1F5.hIgE) or control(SKO-007) IgE. After 24 h, propidium iodide (PI) was added to label deadcells, and the mixture analyzed by flow cytometry. The percentage ofCFSE⁺ cells that were also PI⁺ indicated the fraction of dead/dyingtumor cells present. An increase in tumor cytotoxicity was observed when1F5.hIgE was added, compared to the control antibody (FIG. 2A). Themagnitude of this effect was not augmented by increasing theeffector:target ratio above 2:1.

To investigate the mechanism published assay for antibody-dependent cellphagocytosis (Karagiannis 2003 supra), mast cells were labeled with anantibody to c-kit and the percentage of c-kit+/CFSE+ cells measured inthe presence of specific or control IgE. We did not observe anysignificant IgE dependent phagocytic activity by mast by which CBMCsmight induce tumor cell death, the cell mixtures were incubated with aseries of blocking antibodies, or a rat IgG1 isotype control. Theaddition of anti-TNF decreased tumor cytotoxicity from 24.2±3.5% to14.0±0.3% (FIG. 2B). Slight reductions were seen with the otherantibodies tested, but these differences were not statisticallysignificant.

Eosinophils and IgE-Mediated Tumor Cytotoxicity

Tumor eosinophilia has been associated with a favorable prognosis,especially in tumors of the gastrointestinal tract (Fernandez-Acenero etal., 2000 Cancer 88:1544-1548; Iwasaki et al., 1986 Cancer 58:1321-1327;Pretlow et al., 1983 Cancer Res 43:2997-3000).

Previously, Karagiannis et al. have reported that eosinophils isolatedfrom human peripheral blood mediate cytotoxicity when tested with theovarian cancer cell line IGROV, in an IgE-dependent fashion Karagianniset al., 2007 J Immunol 179:2832-2843). To obtain sufficient numbers ofnaïve human eosinophils, we differentiated cord blood mononuclear cellsin the presence of IL-3 and IL-5. Eosinophils obtained by this protocolexhibit phenotypic and functional similarities to peripheral bloodeosinophils (Zardini et al., 1997 J Immunol Methods 205:1-9). After 3weeks, >95% of live cells in cultures resemble mature eosinophilsphenotypically (CD66b⁺, CD16⁻) as analyzed by flow cytometry. Wedesignated these cells cord blood-derived eosinophils (CBEos). We mixedCBEos with OCI-Ly8 B cells, and added 5.0 μg/ml of either control (SKO)or tumor specific (1F5.hIgE) IgE antibodies. After 24 h at 37° C., PIwas added to label dead cells, and the mixture analyzed by flowcytometry (FIG. 2C). As observed with CBMCs, CBEos triggered increasedtumor cell death in the presence of tumor-specific IgE compared tocontrol IgE. Interestingly, the antibody-dependence of this effect wasless pronounced at higher effector-target ratios, suggesting that highereosinophil to tumor ratios can lead to cell death in anantibody-independent fashion.

To investigate the mechanism of CBEos-mediated tumor cytotoxicity, weincubated the cultures with a panel of blocking antibodies andinhibitors (FIG. 2D). We observed a modest decrease in tumor death withblocking antibodies to TNF-Related Apoptosis Inducing Ligand (TRAIL),and a more significant effect upon addition of a low concentration ofheparin (10 U/ml). Heparin, an anionic molecule, is thought to exertthis effect by neutralizing the cationic proteins released byeosinophils (eosinophil cationic protein (ECP), major basic protein(MBP), eosinophil peroxidase (EPO) and eosinophil derived neurotoxin(EDN)) (Swaminathan et al., 2005 Biochemistry 44:14152-14158). Thesecationic proteins have been shown to cause eukaryotic cell death bydisrupting negatively charged cell membranes (Carreras et al., 2003Biochemistry 42:6636-6644).

Cytokine Profiling of Activated CBMCs

A large number of genes are up-regulated by activated mast cells upontheir activation by IgE and antigen (Sayama et al., 2002 Immunol 3:5).To investigate which cytokines/chemokines are produced by mast cellsactivated by tumor specific IgE, we performed an unbiased screen forcytokines Supernatants from mast cells activated by coculture with IgEand tumor cells were analyzed for 36 cytokines using a multiplexedbead-based assay. CBMCs were activated through FcεRI for 24 hrs at 37°C. by two methods: co-culture with IgE and anti-IgE, or by co-culture ofanti-hCD20 IgE with hCD20-expressing tumor cells. A panel ofinflammatory, growth and chemotactic factors were assessed. To identifycytokines significantly up-regulated in activated versus resting mastcells, we applied a 2-class Significance Analysis of Microarrays (SAM)algorithm (q<0.05, fold change>5.0). SAM identified inflammatorycytokines such as macrophage inflammatory protein (MIP)-1α, MIP-1β,granulocyte macrophage colony stimulating factor (GM-CSF), epithelialneutrophil activating peptide 78 (ENA78) and IL-8. As expected, thestrength of the response was greater when mast cells were activated witha multi-valent antigen (e.g. a cell surface antigen), than whenactivated by simple bivalent crosslinking (IgE+anti-IgE).

Example 3 Anti-MUC1 IgE Antibodies Inhibit In Vivo Tumor Growth

To test the in vivo anti-tumor activity of anti-hMUC1 IgE (3C6.hIgE), wecreated a murine cell line that expressed the transmembrane form ofhMUC1. We transfected the full-length hMUC1 cDNA into the murine breastcarcinoma 4T1. 4T1 was isolated from a spontaneously arising mammarycarcinoma in a Balb/c mouse (Dexter et al., 1978 Cancer Res38:3174-3181) and has been used as a transplantable model of breastcancer (Pulaski et al., 2001 Curr Protoc Immunol Chapter 20:Unit 20 22).4T1.hMUC1 expresses abundant hMUC1 on its cell surface, and growssubcutaneously in hFcεRI transgenic mice with kinetics similar to thoseobserved for the parental 4T1 cell line.

Human FcεRI Mouse Model

To study the in vivo effects of targeting tumors with chimeric human IgEantibodies, we used a human FcεRIα transgenic mouse (hFcεRI Tg⁺). Inthese mice, the endogenous gene encoding the α-subunit of the highaffinity IgE receptor, FcεRIα, has been disrupted, and the mice aretransgenic for the human homologue, under the control of the humanFcεRIα promoter (Dombrowicz et al., 1996 J Immunol 157:1645-1651). Incontrast to wild type mice, where FcεRIα expression is limited to mastcells and basophils, the range of expression of FcεRIα in hFcεRI Tg⁺mice resembles that seen in humans. In addition to mast cells andbasophils, in hFcεRI Tg⁺ mice (and humans) FcεRI is expressed oneosinophils, monocytes, Langerhans cells, B cells and eosinophils(Kinet, J. P. 1999 Annu Rev Immunol 17:931-972; Kayaba et al., 2001 JImmunol 167:995-1003). The hFcεRIα gene product has the capacity tocomplex with the mouse beta and gamma subunits to form a functional 4chain receptor (αβγ₂). hFcεRI Tg⁺ mice mount an anaphylactic response tohuman IgE antibodies and allergen (Dombrowicz et al., 1996 supra).

To verify the ability of these mice to respond to human IgE, weadministered 4T1.hMUC1 tumor cells into the peritoneum, followed byeither control IgE (derived from SKO-007) or anti-hMUC1 human IgE(3C6.hIgE) on day 9. After 24 h, peritoneal lavage was performed, thecells collected, cytospins made, and stained with hematoxylin, eosin andtoluidine blue. Mast cells from the control group were intact, whilethose from the anti-hMUC1 group showed clear evidence of degranulation.This indicates that mast cells from hFcεRI Tg⁺ mice are able to respondto human IgE in an antigen-specific manner.

Tumor-Specific IgE Inhibits In Vivo Tumor Growth

The capacity of hMUC1-specific IgE to affect 4T1.hMUC1 tumor growth invivo was tested in hFcεRI Tg⁺ mice. For these experiments, we consideredthe intraveneous and intraperitoneal delivery of IgE. We found that IgEis rapidly cleared in vivo. This observation along with the fact thatsubcutaneous tumors are not well vascularized, led us to administer thedrug in the peritumoral region.

4T1.hMUC1 tumor cells (a total of 10⁵) were inoculated subcutaneously(s.c.) into the shaved flanks of mice, and treated with 20 μg SKO or3C6.hIgE on days 1, 2, 3, 4 and 5 (FIG. 3A). We observed a modestinhibition of tumor growth in mice treated with 3C6.hIgE (24% reductionin tumor size, p<0.001 by two-way ANOVA). Also, only 3 of 8 mice in thecontrol group survived to day 34, while 5 of 8 mice in the anti-hMUC1group were still alive.

Tumor samples were obtained from surviving mice at day 34, and stainedfor the presence of mast cells using toluidine blue (FIG. 3C). Wedetected the presence of peri- and intra-tumoral mast cells in tumorsfrom mice treated with control IgE. No significant increase in mast cellor inflammatory cell numbers was observed in the 3C6.hIgE treated mice,except for one mouse, which had mast cell infiltration of one tumorsection, accompanied by evidence of mast cell degranulation. Tumorsretained hMUC1 expression even after treatment with anti-hMUC1 IgE(3C6.hIgE), as analyzed by immunohistochemistry.

Enhanced Tumor Responses with Local Delivery of Tumor-Specific IgE andChemokines.

The absence of pronounced anti-tumor responses in our in vivo model maybe due two factors: first, the inability to deliver adequate amounts ofantibody to a poorly vascularized and rapidly growing subcutaneoustumor; and second, the lack of sufficient effector cells in themicroenvironment of these transplanted tumors. To test these variables,we created 4T1 cell lines that produced anti-hMUC1 mouse IgE (mIgE) orchemoattractant cytokines MCP-1 and IL5 (Table 1).

TABLE 1 4T1 derivative cell lines 4T1/ 4T1.hMUC1/ 4T1.hMUC1/ 3C6.mIgEIL-5 MCP-1 Cell surface hMUC1 − + + expression Anti-hMUC1 mouse 1ug/ml^(a) − − IgE production Mouse IL-5 − 10 ng/ml^(b) − productionMouse MCP-1 − − 1 ng/ml^(b) production ^(a)measured in a mouse IgEspecific ELISA assay ^(b)measured at confluence in a mouse cytokinespecific capture ELISA assay

We chose MCP-1/CCL2 because of reports that this cytokine is produced bytumor cells in response to oncogene activation and may be responsiblefor chemotaxis of monocytes and mast cells into the peritumoral stroma(Soucek et al., 2007 Nat Med 13:1211-1218). Interleukin is both a growthfactor and chemotactic factor for eosinophils (Sanderson, C. J. 1988 DevBiol Stand 69:23-29). We mixed combinations of the antigen/cytokineproducing 4T1 cells with mIgE producing 4T1 cells and injected themsubcutaneously in hFcεRI Tg⁺ mice (FIG. 4A). Mixtures of antibodyproducing and antigen expressing tumors grew progressively as did tumorsthat expressed both MCP-1 and IL-5 but lacked local antibody production.In contrast, tumors that expressed anti-hMUC1 mouse IgE, the targetantigen, and both cytokines failed to grow in 7 of 8 mice (FIG. 4B). Inthe single mouse that developed a tumor, it was only visible beginningon day 19 instead of day 8. Eosinophil-containing immune infiltrateswere observed in tumors expressing MCP-1 and IL5. However, tumorelimination was only observed when tumor-specific IgE was also present.These data suggest that when sufficient amounts of tumor specific IgE isdelivered to an antigen-bearing tumor, in the presence of allergiceffector cells, a complete and durable response can be observed.

Interestingly, when wild type 4T1 tumor cells (producing neither hIgEnor chemokines) were injected s.c. into the opposite flank of the 7 micewhich had rejected their tumors, the wild type tumors failed to developafter 30 days (data not shown). These data suggest that IgE+chemokinetransfected tumor cells may be used prophylatically as a tumor vaccine.

To confirm this important observation, this experiment was repeated, andthe results are shown in FIG. 5 (panel C, individual tumors, and panelD, average of the tumor volumes). In the group 4 mice, one tumor wasobserved, which grew with greatly reduced kinetics, compared to theother tumor groups. In this repeat experiment, we observed that micebearing group 3 tumors (antigen+cytokines only) developed overt signs ofmetastases early in the course of tumor growth. We speculated that thetumors implanted in group 3 grew somewhat slower than groups 1 or 2because the mice in which they were growing was sick, and lost weightearly in the course of the experiment.

Example 4 IgE Antibodies Inhibit Tumor Metastases

The experiment described in FIG. 4 of Example 3 was repeated withwild-type mice (Balb/c) instead of the human FcεRIα transgenic micedescribed in Example 3. This was done to assess the contribution of thespectrum of expression of FcεRI in the transgenic mice, compared to thewild type mice, in mediating the observed tumor response.

When the experiment described in example 3 was repeated in wild typemice, we observed a different result. Instead of complete tumorrejection of the Group 4 tumors, as had been seen in the transgenic mice(FIGS. 4B and 5), very small tumors, with greatly reduced growthkinetics, emerged in Group 4 wild-type mice (FIG. 6). Three FcεRIαtransgenic mice were included in this experiment as a positive control.None of three (0/3) Tg+ mice with implanted group 4 tumors showed tumorgrowth (FIG. 6D, □—□).

Even though the Group 4 tumors in wild-type mice grew with greatlyreduced kinetics, they still grew progressively. As we had observed inthe previous experiment with Tg+ mice, the group 3 tumors in wild-typemice developed metastases very early in the course of the experiment.The Group 3 wild-type mice lost weight, developed ascites, and gotruffly fur. However, in the Group 4 wild-type mice, even though theydeveloped small tumors that lagged in growth and appearance from Groups1, 2 and 3, the mice did not develop signs of metastatic disease as seenin mice with group 3 tumors. That is, even though the tumors werepresent under the skin in all of the mice, the effect of the cytokinesMCP-1/IL-5 on tumor behavior (metastases) was completely reversed underthe influence of 3C6-mIgE. The mice with group 4 tumors were followedfor a month, and the mice never developed overt metastases though theyhad very slowly progressively growing tumors on their flanks. Theresults of this key experiment are summarized in Table 2 below.

TABLE 2 Tumor growth Metastasis Tumor Fc-Epsilon Fc-Epsilon GrowthMetastasis Tumor Group transgenic transgenic Wild type Wild type Group1: MUC1 +++++ −−−−− +++++ −−−−−− Group 2: MUC1 +++++ −−−−− +++++ −−−−−−−plus MUC1 IgE Group 3: MUC1, +++ ++ +++ +++++ IL-5 plus MCP-1 Group 4:MUC1, −−−−− −−−− + −−−−−− 3C6-mIgE, IL5, and MCP1 MUC1 = 4T-1/MUC1; MUC1IgE = 4T-1 transfected with the 3C6 mouse heavy and light chain specificfor hMUC1; IL5 = 4T1/hMUC1 transfected with the cDNA for mIL5); MCP-1 =4T1/hMUC1 transfected with the cDNA for MCP-1.

Early metastases was seen in experiments done in both the Tg+ and wildtype mice bearing group 3 tumors. This was completely reversed in micebearing group 4 tumors. The difference between the Tg+ and wild typemice, was that in the Tg+ mice, the mice injected with group 4 tumors,never develop palpable tumors. In the wild type mice, the group 4 tumorsgrow, albeit, slowly. Therefore, the effect on the mIgE antibody has tobe on reprogramming the cells drawn to the tumor by the cytokines, andnot strictly inhibition of tumor growth, per se. A reasonableexplanation of why the mice with group 4 tumor in Tg+ mice do notdevelop metastases is that the tumor is eliminated at the site ofinjection by an allergic immune response. However, in the wild typemice, the metastatic phenotype is eliminated, by mIgE, independent ofthe presence of the tumor. In other words, only when the experiment isconducted in wild type mice, could we observe that the effect of thecytokines on the tumor was reversible (FIG. 6), and that the 3C6-mIgEantibody was able to reprogram the myeloid cells in and around thetumor, likely by preventing tumor engraftment (in the Tg+ mice), orgreatly slowing their growth (WT mice), but in both cases, preventingtumor metastases.

In addition, the experiment conducted with wild-type mice also showsthat the antibody can control an established tumor, as their growthkinetics in the wild type mice are dramatically reduced (FIG. 6).

Therefore, one feature of the IgE antibodies of the invention is theirability to prevent circulating cells from setting up requiredmicroenvironments in the liver, lungs and bones that are necessary priorto the appearance of overt metastases from a primary tumor. IgEantibodies, unlike IgG antibodies, migrate out of the vascular space toevery tissue in the body. Thus IgE antibodies would be uniquely positionto prevent circulating cells from forming a microenvironment necessaryto support metastases. Treatment with such IgE antibodies wouldtherefore prevent metastases and recurrence of cancer.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. It will also be understood that noneof the embodiments described herein are mutually exclusive and may becombined in various ways without departing from the scope of theinvention encompassed by the appended claims.

What is claimed is:
 1. An IgE monoclonal antibody that specificallybinds an epitope of MUC1 and is capable of modulating immune effectorcells to inhibit tumor metastasis having a heavy chain variable regionencoded by a nucleic acid comprising CDRs 1-3 of a heavy chain variableregion encoded by SEQ ID NO: 1 and a light chain variable region encodedby a nucleic acid comprising CDRs 1-3 of a light chain variable regionencoded by SEQ ID NO: 2, optionally humanized.
 2. The antibody of claim1, which has a constant region that is of human origin.
 3. The antibodyof claim 1, which is a chimeric antibody.
 4. The antibody of claim 1,which is a humanized antibody.
 5. The antibody of claim 1, which is3C6.hIgE.
 6. A method for inhibiting metastasis of a solid tumor in apatient, comprising the step of administering to the patient theantibody of claim
 1. 7. The method of claim 6, wherein the antibody hasa constant region that is of human origin.
 8. The method of claim 6,wherein the antibody is a chimeric antibody.
 9. The method of claim 6,wherein the antibody is a humanized antibody.
 10. The method of claim 6,wherein the antibody is 3C6.hIgE.
 11. The method of claim 6, wherein theantibody is administered to the patient prior to the development ofmetastasis in the patient.
 12. The method of claim 6, wherein the solidtumor is of epithelial origin.
 13. The method of claim 6, wherein thesolid tumor is breast cancer, colorectal cancer, ovarian cancer, renalcancer, prostate cancer, bladder cancer, gastrointestinal cancer,pancreatic cancer, or lung cancer.